实时荧光定量环介导等温扩增方法检测马铃薯黑胫病菌

正由马铃薯黑胫果胶杆菌Pectobacterium atrosepticum引起的生产上日趋严重的马铃薯黑胫病,其初侵染来源是带菌种薯(Mattinen et al.,2008),种薯带菌数量直接决定田间病害发生的严重程度。因此,播种前明确种薯中马铃薯黑胫果胶杆菌的含量至关重要。目前国内外应用实时荧光定量PCR方法对病原菌进行定量检测已有很多(娄兵海等,2014),但还未见利用实时荧光定量环介导等温扩增     

Simple and sensitive BIO-PCR detection of potato blackleg pathogens from stem,tuber, and soil samples

Journal of General Plant Pathology - A BIO-PCR was developed for the detection of potato blackleg pathogens, including Pectobacterium wasabiae (Pw), P. atrosepticum (Pa), P. carotovorum subsp....     

Etiology of potato soft rot and blackleg diseases complex in Egypt

Journal of Plant Diseases and Protection - Recent observations of increasing incidences of potato plants showing blackleg symptoms have caused serious concern for potato growers and traders in...     

Detection,quantification and classification of soft rot erwinias associated with potato tubers

An improved method for detection, quantification and classification of soft rot bacteria associated with potato seed tubers, plant material, soil and water has been developed. The method is based on the use of a modified version of the crystal violet pectate selective medium (CVP), enrichment cultures under anaerobic conditions using pectate as the sole carbon source for recovery improvement, and the quantitative estimation ofErwinia spp. by employing a new solid medium - most probable number (MPN) method. The use of this method enabled an improvement in the recovery and identification of specificErwinia spp. in mixed populations. This was done by incubating CVP plates — used for the MPN counting — at three different temperatures (15, 28 and 39°C). These combined techniques were used for estimating low level populations at less than one cell per gram or ml tested ofErwinia carotovora subsp.carotovora, E. carotovora subsp.atroseptica, andE. chrysanthemi.     

Microbiological, immunological and molecular methods suitable for commercial detection and quantification of the blackleg pathogen, Erwinia carotovora subsp. atroseptica, on seed potato tubers: a review

Contamination of seed potato tubers by Erwinia carotovora subsp. atroseptica is widespread with the bacteria usually sited superficially in lenticels and suberized wounds. As seed contamination level is related to blackleg incidence, seed health is best assessed by determining the number of cells of E. c. atroseptica per mL of tuber-peel extract. The relative specificity, sensitivity and ease of use of four recently developed microbiological, immunological and molecular methods to detect and/or quantify tuber contamination are discussed in relation to the testing of commercial seed stock. Sensitivities of all four methods are at or below the threshold level for blackleg development (< 10³ cells mL^-1), but there are differences regarding their specificity and ease of use. Three of them allow enumeration of most live cells of the bacterium, using specific monoclonal and polyclonal antibodies against the predominant serogroup I: (a) immunomagnetic separation of E. c. atroseptica before viable count on a selective-diagnostic growth medium, crystal violet pectate, (b) immunofluorescence staining and counting of colonies in pour-plate medium in tissue culture plates and (c) enrichment of the bacterium in peel-extract dilutions directly in microtitre plates prior to DAS-ELISA. In the fourth method, both live and dead cells are detected, but not quantified, by PCR amplification of target sequences using specific primers for E. c. atroseptica regardless of serogroup.     

Methods of testing potato cultivars for resistance to soft rot of tubers caused by Erwinia carotovora subsp. atroseptica

The resistance of eight potato cultivars to tuber soft rot caused by E. carotovora subsp. atroseptica was assessed in 2 years using three different test methods. Similar cultivar resistance rankings were obtained for any one method within a year and between years for two methods (single site, infectivity titration), but not for the third (vacuum infiltration). However, the ranking of cultivars differed for the three methods. Ranking was not affected by inoculating the cortex or the more susceptible medullary tissue, or by assessing rotting in terms of infection frequency or lesion size, but it was affected by oxygen concentration during incubation. Differences among cultivars were greater when inoculated tubers were incubated anaerobically than when incubated with 5% oxygen. There was no relationship between the relative susceptibilities of cultivars to tuber soft rot in storage in January/February and those of mother tubers after planting.     

Stem and bulb nematode, Ditylenchus dipsaci, associated with a dry rot of potato tubers

About 10% of the ware-sized tubers from a 1990 potato crop were found to have dry rot. The affected tubers had well defined circular patches of dry rot, either with the tuber surface depressed and cracked, or with deep, open cavities where the tuber surface had disintegrated. The rotted tissues contained large numbers of the stem and bulb nematode, Ditylenchus dipsaci. This is the first recorded case in the UK; dry rot of potato tubers is usually caused by the potato tuber nematode D. destructor.     

河北和内蒙古马铃薯干腐病菌种类鉴定

为明确河北和内蒙古马铃薯干腐病菌种类和优势种群,从两省采集300块具干腐病症状的样品,通过分离培养、形态鉴定、TEF-1α序列分析及致病性测定等,对马铃薯干腐病菌种类进行了系统研究.结果表明,两省马铃薯干腐病共存在4种病原菌,即接骨木镰刀菌Fusarium sambucinum、锐顶镰刀菌F.acuminatum、尖孢镰刀菌F.oxysporum和芬芳镰刀菌F.redolens,分别为149、80、3、3株.其中,河北省的优势种群为接骨木镰刀菌和锐顶镰刀菌,其发生频率分别为50％和48.68％;内蒙古的优势种群为接骨木镰刀菌,发生频率为87.95％.     

马铃薯环腐病生防菌株P1的鉴定、防病效果及促生作用研究

 在马铃薯环腐病区采集健康的马铃薯Solanum tuberosum块茎,从中分离到1株对马铃薯环腐病菌Clavibacter michiganense subsp. sepedonicum具有强拮抗作用的内生细菌P1。经形态观察、生理生化鉴定,菌株P1归属为Bacillus sp.,进一步经16SrDNA序列对比分析,确定为巨大芽孢杆菌B. megatherium。经证明,P1菌株的培养液抗菌粗提物属于蛋白类,该抗菌蛋白对紫外光不敏感,pH为7.0时抑菌活性最强,温度高于80℃时抑菌活性明显下降。温室试验表明,P1菌株能显著提高马铃薯植株的株高、茎粗、产量及大薯率,其对马铃薯环腐病防效达53.4%。     

Enzyme-linked immunosorbent assay (ELISA) for the detection of potato viruses S and M in potato tubers

Enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase successfully detected potato virus S (PVS) and potato virus M (PVM) in secondarily infected tubers of some Dutch potato cultivars. Extinction was higher for PVS than for PVM but values for both declined slightly within 8 weeks of lifting and it is suggested that testing be carried out within this period. Values (A405) of ELISA reactions between healthy and infected tubers were statistically significant and storage at 4° or 20°C had no effect on detectability of the viruses.Samenvatting Aardappelvirus S kon zowel in knollen van oogst 1978, die gedurende 49 weken bij 4°C waren bewaard en waarvan de kiemrust reeds was verbroken, als in knollen van oogst 1979, die bij 4° of 20°C waren bewaard en nog in de kiemrust verkeerden, tot 8 weken na rooien betrouwbaar met ELISA worden aangetoond. Aardappelvirus M kon eveneens met ELISA betrouwbaar worden aangetoond in knollen van oogst 1979, bewaard bij 4° of 20°C, tot 8 weken na het rooien.De extinctiewaarden voor aardappelvirus S waren hoger dan die voor aardappelvirus M. De waarden voor beide virussen vertoonden een daling gedurende de onderzoekperiode (tot 8 weken na rooien). Er kon geen effect van de bewaartemperatuur (4° en 20°C) op de aantoonbaarheid van de virussen worden aangetoond. Geen verschillen werden waargenomen tussen de extinctiewaarden van het sap uit navel- en krooneinden van de knollen, die nog in de kiemrust verkeerden.Guest worker from April–September 1979 as a fellow of the International Agricultural Centre, Wageningen, the Netherlands, from the Agricultural Research Centre, Yanco, NSW 2703, Australia.     

Occurrence,identification, and host range of Pectobacterium brasiliense causing soft rot on seed potato tubers in Turkey

Journal of Plant Diseases and Protection - Pectobacterium brasiliense is an important bacterial species that causes soft rot disease in a wide range of host plants including potatoes worldwide. In...     

Heat treatment of seed tubers for control of potato blackleg (Erwinia carotovora subsp. atroseptica) and other diseases

The thermal death points of Erwinia carotovora subsp. atroseptica and subsp. carotovora were determined in relation to duration of heat treatment, age of culture and culture medium. No isolates cultured in liquid media survived heating at 53°C for 5 min while those on solid media were killed by heating at 54°C for 10 min. After immersing naturally contaminated potato tubers for 10 min in water at 55°C, Erwinia could not be detected. The same treatment of naturally or artificially contaminated seed tubers gave complete absence of blackleg infection in the field and decreased the amounts of powdery scah(Spongospora subterranea) and black scurf (Rhizoctonia solani) on progeny tubers.     

甘肃省马铃薯镰刀菌干腐病优势病原的分离鉴定

 为明确甘肃马铃薯镰刀菌干腐病的优势病原,2006 年12 月~ 2007 年3 月由西至东从甘肃张掖、天祝、永登、临洮、渭源和西和等6 县市的马铃薯贮藏窖中采集表现有镰刀菌干腐病症状的马铃薯薯块,以组织分离法分离病原,单孢纯化镰刀菌(Fusarium spp. )菌株后,以形态学为基础,参照Nelson 镰刀菌分类系统进行鉴定。结果表明:6 个采样区共分离到293株镰刀菌菌株,其中以接骨木镰刀菌(F. sambucinum)和茄病镰刀菌(F. solani)出现频率高,是优势种。分析发现第一优势种随采样区而异,张掖、天祝和渭源采集的样品中茄病镰刀菌分离频率分别为42. 6% 、42. 1% 和32. 4% ,接骨木镰刀菌分离频率分别为14. 8% 、5. 3% 和26. 5% ,茄病镰刀菌为第一优势种;永登、临洮和西和采集的样品中接骨木镰刀菌分离频率分别为52郾1% 、50. 9% 和55. 2% ,茄病镰刀菌的分离频率分别为23. 3% 、32. 7% 和20. 7% ,接骨木镰刀菌为第一优势种。本文进一步对其在PDA、CLA 上的培养特征进行了观察和描述。按照柯赫氏法则用混合菌株接种法对大西洋(Atlantic)、夏波蒂(Shepody)以及一地方品种进行致病性测定,证实了优势菌种对块茎的致病性。利用EF鄄1琢基因引物(EF鄄1H 和EF鄄2T)对接骨木镰刀菌菌株GAUF鄄F12 进行基因组DNA 的PCR 扩增,将PCR 产物回收测序后在GenBank 上比对,菌株GAUF鄄F12 与GenBank 登记的接骨木镰刀菌5 个菌株的同源性均达99% ;用DNASTAR 分析软件将同源性较高的登记菌株的序列与GAUF鄄F12 菌株构建同源性树,结果表明:该菌株与以上5 个接骨木镰刀菌菌株均位于同源性树的同一分支,聚为一类,与形态学的鉴定结果一致。     

The distribution of potato spindle tuber viroid in potato plants and tubers1

Potato spindle tuber viroid (PSTV) in potato plants was investigated by ‘return’ gel electrophoresis. The experiments were carried out under quarantine conditions in the greenhouse with primarily and secondarily infected plants. The PSTV content in different plant parts was estimated by the intensity of the viroid band in polyacrylamide gel. The results showed a decrease of viroid content from the upper to the lower parts of the plant. In both primarily and secondarily infected plants, PSTV was reliably detected in the top leaves, but less so in the lower leaves. In four out of ten secondarily infected plants, PSTV was found in the roots. In dormant tubers, the bands were more intense with samples obtained from the rose end and the heel than from those obtained from the medullary tissue. With one exception, all 64 tubers from 26 primarily infected plants were infected with PSTV.     

Epidemiology of bacterial ring rot of potato in plant and soil–consequences for disease management

Journal of Plant Diseases and Protection - In five field experiments, carried out from 2002 to 2004, the infestation of potato plants and the contamination of soil with Clavibacter michiganensis...     

Investigation and control of potato brown rot in Germany,especially in Bayern1

Ralstonia solanacearum has been found in Dutch potato production since 1995. For this reason, comprehensive laboratory tests on seed- and ware-potato production have been carried out in Germany. The amount of tested potato lots has increased fivefold. In 1997, 10678 tests were carried out on seed potatoes and 2351 tests on ware potatoes respectively. In 1996, R. solanacearum was found in five lots in Bayern and in one lot in Brandenburg, and in 1997 only in two ware-potato lots in Bayern. A thorough analysis was carried out to find the source of infection, but it was not possible to identify it clearly. The measures taken were based on the draft Control Directive for brown rot, which will soon be adopted by the Council of the European Union, and were very restrictive for the affected farms. Monitoring of a potato-processing enterprise that had processed an infected lot without permit in 1996 established that R. solanacearum was present in wastewater. This paper discusses the measures taken in Bayern and the risks of further introduction.     

Endophytic bacterial communities in the periderm of potato tubers and their potential to improve resistance to soil-borne plant pathogens

To evaluate whether the location of bacterial endophyte communities contributes to disease resistance in potato tubers ( Solanum tuberosum ), the population density, biodiversity and antibiotic activity of endophytic bacteria was examined from the tuber peel (periderm plus top 3 mm of tissue) of four cultivars (Russet Burbank, Kennebec, Butte and Shepody). There were no significant differences for population density of bacteria among the layers of peel examined and no cultivar × peel layer interaction. Endophytic bacteria from several layers of peel were challenged in in vitro bioassays to the soil-borne plant pathogens Fusarium sambucinum , Fusarium avenaceum , Fusarium oxysporum and Phytophthora infestans (mating types A1 and A2). In general, antibiosis of bacterial endophytes against these pathogens was significantly higher ( P  = 0.01) in isolates recovered from the outermost layer of tuber peel and decreased progressively toward the centre of the tuber. Antibiosis against P. infestans was variable, with a progressive decrease in antibiotic activity from outer to inner layers of peel occurring in cvs Russet Burbank and Kennebec only. For antibiosis there were significant cultivar × peel, and cultivar × pathogen interactions ( P  = 0.01). In all cases the inhibitory activity of endophytic bacteria was significantly greater ( P  = 0.01) against the A1 than the A2 mating type of P. infestans . In four of seven cases, where the same species of bacteria were recovered from all three peel layers, antibiosis to pathogens decreased significantly ( P  = 0.01) with depth of recovery (from the periderm to inside the tuber), indicating that in certain communities of endophytic bacteria, defence against pathogens may be related to bacterial adaptation to location within a host and may be tissue-type and tissue-site specific.     

内蒙古马铃薯黑胫病病原菌的分离和鉴定

 马铃薯是我国的第四大主粮。内蒙古是我国最大的马铃薯生产基地,马铃薯产业是当地农民收入的主要来源。2017年8月,内蒙古锡林郭勒盟部分马铃薯田块发生了一种细菌性黑胫病,发病面积约为200亩,发病率约为20%。发病初期,植株的茎基部变色、发黑,伴有臭味,严重时茎秆腐烂,植株死亡。为明确该地区马铃薯细菌性黑胫病的病原菌种类,本研究对该病原菌进行了分离,并利用生理生化测定,Biolog分析和分子生物学手段将该病原菌鉴定为胡萝卜软腐果胶杆菌巴西亚种(Pectobacterium carotovorum subsp. brasiliense)。据以往报道,我国马铃薯黑胫病主要由黑腐果胶杆菌(P. atrosepticum)和胡萝卜软腐果胶杆菌胡萝卜亚种(P. carotovorum subsp. carotovorum)两种病原菌引起,本研究是P. carotovorum subsp. brasiliense引起马铃薯黑胫病在国内的首次报道。     

Test performance study for detection of Ralstonia solanacearum and Clavibacter sepedonicus in potato tubers with TaqMan PCR

A test performance study (TPS) was organized in 2018 with ten official testing laboratories to evaluate the performance of different real-time PCR tests for the detection of Clavibacter sepedonicus and/or Ralstonia solanacearum in potato tubers. Participants were sent spiked potato extracts with low (0.8–1.2 × 10⁴ cfu mL^-1), medium (1.6–2.4 × 10⁵ cfu mL^-1) and high (1.6–2.4 × 10⁷ cfu mL^-1) bacterial loads, DNA extracts thereof and heel-end cores from symptomatic potato tubers. The four real-time PCR tests in this TPS for detection of C. sepedonicus were considered fit for purpose as principal screening methods. Two real-time PCRs in this TPS were considered fit for purpose as principal screening methods for detection of R. solanacearum. A third real-time PCR missed 23% of the DNA samples from low-level R. solanacearum spikes and is considered not fit for purpose as a principal screening method. Correct identification of spiked samples was lower when DNA extraction from the spiked samples was performed by the participating laboratories, highlighting the importance of appropriate DNA extraction protocols.     

梨成熟果实对炭疽病菌和轮纹病菌抗扩展能力的评价

对101个梨品种成熟果实抗炭疽病菌和轮纹病菌扩展能力进行评价、聚类分析,并对其抗病菌扩展能力与生理指标作相关分析.聚类分析结果表明,101个梨品种果实对两种病菌抗扩展能力均可分为五类即抗扩展性强、较强、中等、较弱、弱.其中对炭疽病菌和轮纹病菌抗扩展性中等的品种最多,分别占供试品种40.6％和35.6％,而对这两种病菌抗扩展性弱的品种最少,分别占供试品种5.9％和2.0％.相关分析表明,果肉总酚含量分别与果实抗炭疽病菌和轮纹病菌的扩展能力呈极显著和显著正相关;果肉石细胞含量与果实对这两种病菌抗扩展的能力均呈正相关,其中与轮纹病达到极显著水平;而果肉木质素含量和果实对这两种病菌抗扩展能力之间相关性不明显.